This preprint is under consideration at Journal of Neuroinflammation. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
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Research
Lesley Probert
Hellenic Pasteur Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5931-2880
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This work is licensed under a CC BY 4.0 License. Read Full License
BACKGROUND
During inflammatory demyelination TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF, whereas TNFR2 mediates beneficial effects of transmembrane TNF through oligodendrocytes, microglia, and possibly other cell types. This model supports use of selective inhibitors of soluble TNF/TNFR1 as antinflammatory drugs for CNS disease. A potential obstacle is the neuroprotective effect of soluble TNF pretreatment described in cultured neurons, but the in vivo relevance is unknown.
METHODS
To address this question we generated mice with neuron-specific depletion of TNFR1, TNFR2 or IKKβ and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of soluble TNF (and therefore TNFR1) neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wildtype and TNF and TNF receptor knockout cells and measuring NMDA excitotoxicity in vitro.
RESULTS
Neither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKβ promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid excitotoxicity model in mice, and limited hippocampal neuron death. The neuroprotective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. Here as expected, pretreatment of astrocyte-neuron co-cultures with soluble TNF protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte transmembrane TNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist.
CONCLUSIONS
These results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKβ promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously-described neuroprotective effects of soluble TNF (and therefore TNFR1) against glutamate excitotoxicity in vitro are indirect, and mediated by astrocyte transmembrane TNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of soluble TNF/TNFR1 with maintenance of TNFR2 function would have anti-inflammatory and neuroprotective properties required for the safe treatment of CNS disease.
Keywords
neuroinflammation, immunotherapy, multiple sclerosis, NF-κB, autophagy, neuroprotection, neurodegeneration, oligodendrocyte death, glutamate excitotoxicity, TNFR2 agonist
Figure 1
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Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.jpg
Brain-specific depletion of TNFR1 and TNFR2 in tissues of nTNFR1KO and nTNFR2KO mice. Allele-specific DNA PCR analysis was used to assess the tissue specificity of Cre-mediated recombination (deletion) events in different tissues of LoxP-flanked (“floxed”) Tnfrsf1a (A) or Tnfrsf1b (B) sequences in mice (“defloxed”). Deletion of floxed Tnfrsf1a and Tnfrsf1b alleles was restricted to brain (cortex, hippocampus and cerebellum) and spinal cord of nTNFR1KO and nTNFR2KO mice, and not TNFR1ff or TNFR2ff control mice, respectively. Control tissues are brain from nTNFR1KO (A; Cre ff), nTNFR2KO (B; Cre ff) and WT B6 (+/+) mice.
- SupplementaryFigure2.jpg
Neuronal TNFR2 does not affect cuprizone demyelination and remyelination. (A) Semiquantitative scoring of demyelination (loss of LFB staining) in the corpus callosum of nTNFR2KO and TNFR2ff control naive (CPZ0) or CPZ-fed CPZ5 and CPZ6+2 mice. Quantitative representation of (B) CNPase immunoreactivity by densitometry, (C) Iba1 immunoreactivity by % area covered, and (D) numbers of APP immunoreactive spheroids/mm2 tissue in the corpus callosum in serial coronal paraffin sections of brain from nTNFR2KO and TNFR2ff control mice. Results are means of 2 (CPZ0; D, CPZ6+2) or ≥5 mice (for all other time points) from one representative of two independent experiments. Statistical significance after comparisons are shown by two-way ANOVA with Bonferroni’s test. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001.
- SupplementaryFigure3.jpg
Neuronal IKKβ contributes to resolution of CNS T cell infiltration during cuprizone remyelination. (A and B) Differential expression of the myelin markers Mbp and Olig2 relative to GusB in total mRNA isolates isolated from nIKKβKO and control (IKKβff) brains from CPZ0 or CPZ2, CPZ3, CPZ5 and CPZ6+1 mice. (Ci) CD3 immunostaining of T cells in serial brain coronal paraffin sections from nIKKβKO and control mice during CPZ demyelination and remyelination. Scale bars: 100 μM. (Cii) Numbers of CD3-immunoreactive T cells/mm2 tissue counted in coronal paraffin sections through the corpus callosum of brain from nIKKβKO and control mice represented in Ci. (D) Quantitative representation of neurofilament H phosphorylated (SMI 31) immunoreactivity in the corpus callosum of nIKKβKO and control mice by densitometry. Results are means of 2 (CPZ6+1) or 3-5 mice from one representative of two independent experiments. Statistical significance after comparisons are shown by two-way ANOVA with Bonferroni’s test (A and B) or Student’s t-test (Cii). * p ≤ 0.05, ** p ≤ 0.005.
- SupplementaryFigure4.jpg
Preconditioning of astrocyte-neuron co-cultures with solTNF provides neuroprotection against NMDA excitotoxicity. (A) Neuron-astrocyte co-cultures at day in vitro 7 (NA-DIV7) were incubated with 100 ng/ml human (h) or mouse (m) TNF for 24 h, excitotoxic death was induced by addition of 50 μΜ NMDA/ 10 μΜ glycine on NA-DIV8 and death was measured after 22 h (NA-DIV9) by Hoechst staining. (B) Images from astrocyte-neuron co-cultures stained with Hoechst, anti-neuronal nuclei (NeuN) for post-mitotic neurons, anti-glial fibrillary acidic protein (GFAP) for astrocytes and their overlay. Scale bar: 20 μM. Results shown are means ± SEM of triplicate samples from one representative of five independent experiments. Statistical significance after pairwise comparisons are shown by Student’s t-test. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001.
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This preprint is under consideration at Journal of Neuroinflammation. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Lesley Probert
Hellenic Pasteur Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5931-2880
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 05 Jan, 2021
No community comments so far
Editorial decision: Minor Revision
On 14 Feb, 2021
Review #2 received
Received 13 Feb, 2021
Reviewer #2 agreed
On 19 Jan, 2021
Review #1 received
Received 07 Jan, 2021
Reviewer #1 agreed
On 30 Dec, 2020
Reviewers invited
Invitations sent on 28 Dec, 2020
Editor assigned
On 25 Dec, 2020
Submission checks complete
On 25 Dec, 2020
Editor invited
On 25 Dec, 2020
First submitted
On 22 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Neurobiology of Disease
License:
This work is licensed under a CC BY 4.0 License. Read Full License
BACKGROUND
During inflammatory demyelination TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF, whereas TNFR2 mediates beneficial effects of transmembrane TNF through oligodendrocytes, microglia, and possibly other cell types. This model supports use of selective inhibitors of soluble TNF/TNFR1 as antinflammatory drugs for CNS disease. A potential obstacle is the neuroprotective effect of soluble TNF pretreatment described in cultured neurons, but the in vivo relevance is unknown.
METHODS
To address this question we generated mice with neuron-specific depletion of TNFR1, TNFR2 or IKKβ and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of soluble TNF (and therefore TNFR1) neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wildtype and TNF and TNF receptor knockout cells and measuring NMDA excitotoxicity in vitro.
RESULTS
Neither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKβ promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid excitotoxicity model in mice, and limited hippocampal neuron death. The neuroprotective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. Here as expected, pretreatment of astrocyte-neuron co-cultures with soluble TNF protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte transmembrane TNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist.
CONCLUSIONS
These results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKβ promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously-described neuroprotective effects of soluble TNF (and therefore TNFR1) against glutamate excitotoxicity in vitro are indirect, and mediated by astrocyte transmembrane TNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of soluble TNF/TNFR1 with maintenance of TNFR2 function would have anti-inflammatory and neuroprotective properties required for the safe treatment of CNS disease.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.jpg
Brain-specific depletion of TNFR1 and TNFR2 in tissues of nTNFR1KO and nTNFR2KO mice. Allele-specific DNA PCR analysis was used to assess the tissue specificity of Cre-mediated recombination (deletion) events in different tissues of LoxP-flanked (“floxed”) Tnfrsf1a (A) or Tnfrsf1b (B) sequences in mice (“defloxed”). Deletion of floxed Tnfrsf1a and Tnfrsf1b alleles was restricted to brain (cortex, hippocampus and cerebellum) and spinal cord of nTNFR1KO and nTNFR2KO mice, and not TNFR1ff or TNFR2ff control mice, respectively. Control tissues are brain from nTNFR1KO (A; Cre ff), nTNFR2KO (B; Cre ff) and WT B6 (+/+) mice.
- SupplementaryFigure2.jpg
Neuronal TNFR2 does not affect cuprizone demyelination and remyelination. (A) Semiquantitative scoring of demyelination (loss of LFB staining) in the corpus callosum of nTNFR2KO and TNFR2ff control naive (CPZ0) or CPZ-fed CPZ5 and CPZ6+2 mice. Quantitative representation of (B) CNPase immunoreactivity by densitometry, (C) Iba1 immunoreactivity by % area covered, and (D) numbers of APP immunoreactive spheroids/mm2 tissue in the corpus callosum in serial coronal paraffin sections of brain from nTNFR2KO and TNFR2ff control mice. Results are means of 2 (CPZ0; D, CPZ6+2) or ≥5 mice (for all other time points) from one representative of two independent experiments. Statistical significance after comparisons are shown by two-way ANOVA with Bonferroni’s test. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001.
- SupplementaryFigure3.jpg
Neuronal IKKβ contributes to resolution of CNS T cell infiltration during cuprizone remyelination. (A and B) Differential expression of the myelin markers Mbp and Olig2 relative to GusB in total mRNA isolates isolated from nIKKβKO and control (IKKβff) brains from CPZ0 or CPZ2, CPZ3, CPZ5 and CPZ6+1 mice. (Ci) CD3 immunostaining of T cells in serial brain coronal paraffin sections from nIKKβKO and control mice during CPZ demyelination and remyelination. Scale bars: 100 μM. (Cii) Numbers of CD3-immunoreactive T cells/mm2 tissue counted in coronal paraffin sections through the corpus callosum of brain from nIKKβKO and control mice represented in Ci. (D) Quantitative representation of neurofilament H phosphorylated (SMI 31) immunoreactivity in the corpus callosum of nIKKβKO and control mice by densitometry. Results are means of 2 (CPZ6+1) or 3-5 mice from one representative of two independent experiments. Statistical significance after comparisons are shown by two-way ANOVA with Bonferroni’s test (A and B) or Student’s t-test (Cii). * p ≤ 0.05, ** p ≤ 0.005.
- SupplementaryFigure4.jpg
Preconditioning of astrocyte-neuron co-cultures with solTNF provides neuroprotection against NMDA excitotoxicity. (A) Neuron-astrocyte co-cultures at day in vitro 7 (NA-DIV7) were incubated with 100 ng/ml human (h) or mouse (m) TNF for 24 h, excitotoxic death was induced by addition of 50 μΜ NMDA/ 10 μΜ glycine on NA-DIV8 and death was measured after 22 h (NA-DIV9) by Hoechst staining. (B) Images from astrocyte-neuron co-cultures stained with Hoechst, anti-neuronal nuclei (NeuN) for post-mitotic neurons, anti-glial fibrillary acidic protein (GFAP) for astrocytes and their overlay. Scale bar: 20 μM. Results shown are means ± SEM of triplicate samples from one representative of five independent experiments. Statistical significance after pairwise comparisons are shown by Student’s t-test. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001.
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